CN110655579B - Novel anti-CT L A-4 monoclonal antibody and application thereof - Google Patents
Novel anti-CT L A-4 monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and particularly provides a novel anti-CT L A-4 monoclonal antibody which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8, and the amino acid sequence of the light chain variable region is SEQ ID NO 9 or SEQ ID NO 10.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a novel anti-CT L A-4 monoclonal antibody and application thereof.
Background
Immunomodulation plays an extremely important role in the immune response of the body, while activation of immunocompetent cells is critical to the regulation of the overall immune response. Studies have shown that T cell activation and proliferation are dependent on dual signaling pathways. The concept of "co-stimulatory signals" was based on the two-signal model of T cell activation in 1970 by Bretscher and Cohn, i.e., T cell activation requires not only presentation of MHC-antigen peptide complexes by APCs to antigen-specific T cells to provide a first signal, but also participation of multiple co-stimulatory molecules in providing a secondary signal (i.e., co-stimulatory signal); with the progress of research, the costimulatory signal gradually becomes a hot spot field of immunology; these costimulatory signaling molecules include mainly two superfamily, CD28/B7 and TNFR/TNF. Homologous B7 family members B7-1 (also known as B7, B7.1 or CD80) and B7-2 (also known as B7.2 or CD86), both of which can deliver a costimulatory signal upon binding to the CD28 antigen on T cells, leading to T cell activation. CD28 is a homodimeric glycoprotein member of the immunoglobulin (Ig) superfamily, which has a single extracellular variable region and which is present on most mature human T cells.
The cytotoxic T lymphocyte-associated antigen-4 (also known as CD152 for short as CT L A-4) is a homolog of CD28 CT L A-4 was discovered in 1987 and has a close relationship with the CD28 molecule in terms of gene structure, chromosomal localization, sequence homology and gene expression, and is a receptor for the costimulatory molecule B7, and is mainly expressed on the surface of activated T cells, however, as a costimulatory signal for lymphocyte activation, CT L A-4 and CD28 molecule function in opposition, CT L A-4 functions primarily to inhibit T cell activation, as demonstrated by CT L A-4 having large-scale lymphoproliferative tissue in deficient mice, studies have shown that CT L A-4 neutralizing antibodies or CT L A-4 proteins can block L A-4 from binding to its natural ligands, thereby blocking the negative T L A-4 and modulating T cell signaling, and thus enhancing the negative response of CT 2 to various human immune related diseases, such as immune response to CT 632-mediated diseases.
In addition, in vitro experiments, the anti-CT L A-4 monoclonal antibody can specifically relieve the immunosuppression of CT L A-4 antigen on organisms, activate T cells, induce I L-2 production, and I L-2 is an important factor for regulating immune response, can promote the proliferation and differentiation of B cells, and participates in antibody reaction, hematopoiesis and tumor monitoring, so that the research of the anti-CT L A-4 monoclonal antibody has wide application prospects in anti-tumor and immune diseases and has huge development potential.
There have been many studies on the anti-CT L A-4 monoclonal antibody, but only Iplilimumab developed by Bristol-Myers Squibb (BMS) was approved by the drug administration departments such as the United states Food and Drug Administration (FDA), European drug administration (EMA), and the Japan pharmaceutical and medical device Integrated agency (PMDA) and sold on the market under the trade name of Yervoy, and the drug approved indication is melanoma, and meanwhile, Bai Shi Guibao applied for an invention patent (CN201210037538.0) under the patent name of Human CT L A-4 antibiodes and their usages at 8/24/2000.
At present, the fully human antibody is the main direction of development of therapeutic antibodies, and the emergence of antibody library technology provides a good technical platform for preparation and screening of the fully human antibody. The antibody library technology bypasses the hybridoma process necessary in the previous monoclonal antibody development process, and can obtain various antibody genes and antibody molecular fragments even without an immunization process. Phage antibody libraries were the earliest and most widely used antibody libraries at present. The phage antibody library display technology is a technology firstly established by Smith, inserts a gene coding a foreign protein or polypeptide into a phage capsid protein gene, and leads the foreign protein or polypeptide and the phage capsid protein to be fused and expressed on the surface of a phage. The phage antibody library utilizes the principle to express antibodies with different specificities or functional fragments thereof (Fab, Fv and ScFv) on the surface of phage, and then carries out screening by using antigen. The phage antibody library is divided into immune library and non-immune library according to the source of antibody gene, and the non-immune library comprises natural library, semi-synthetic library and fully-synthetic library. Screening of phage antibody libraries mimics the process of antibody affinity maturation, typically by coating the antigen on a solid phase medium, adding the phage antibody library to be screened, and performing several rounds of "adsorption-washing-elution-amplification" (i.e., panning) until specific, high affinity antibodies are screened.
Disclosure of Invention
In order to meet the requirements of domestic markets, the invention utilizes a phage antibody library display technology and a high-throughput screening method to screen the anti-CT L A-4 fully human monoclonal antibody with higher affinity and better activity.
The specific technical scheme of the invention is as follows:
the invention provides a novel anti-CT L A-4 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8, and the amino acid sequence of the light chain variable region is SEQ ID NO 9 or SEQ ID NO 10.
Further, the monoclonal antibody is selected from any one of the following:
(N-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10.
Further, the monoclonal antibody also comprises a heavy chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG 4;
preferably, the heavy chain constant region is human IgG 1.
Further, the monoclonal antibody is a full-length antibody or an antibody fragment, and the antibody fragment comprises one or a combination of more of Fab, F (ab)2, Fv or ScFv. The ScFv is a single-chain antibody (single-chain).
Further, the monoclonal antibody is a fully human antibody.
The invention also provides a polypeptide or protein, and the polypeptide or the protein comprises the novel anti-CT L A-4 monoclonal antibody.
The invention also provides a polynucleotide sequence or a combination, wherein the polynucleotide sequence or the combination codes the amino acid sequence of the novel anti-CT L A-4 monoclonal antibody.
The invention also provides a recombinant DNA expression vector which comprises the polynucleotide sequence or the combination.
The invention also provides a host cell for transfecting the recombinant DNA expression vector, wherein the host cell comprises a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell;
preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
The HEK293E cells are human embryonic kidney293E cells (human embryo kidney293E cells); the CHO cell is a Chinese hamster ovary cell (Chinese hamster ovary cell); NS0 cells were mouse NS0 thymoma cells.
The invention further provides a medicine or a medicine composition, which comprises the novel anti-CT L A-4 monoclonal antibody.
The invention further provides application of the novel anti-CT L A-4 monoclonal antibody in preparing a medicament for treating cancer diseases;
preferably, the cancer disease comprises melanoma, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer or liver cancer.
The anti-CT L A-4 monoclonal antibody has strong affinity and good biological activity, can be specifically combined with a CT L A-4 antigen, enhances the activity of T cells, and is further used for treating cancer diseases, wherein the cancer diseases comprise but are not limited to melanoma, prostatic cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer and the like, and therefore the anti-CT L A-4 monoclonal antibody has a wide development prospect.
Drawings
FIG. 1 is a plasmid map of pScFvDisb-s in the antibody biopanning method provided in example 1 of the present invention;
FIG. 2 is a graph showing the comparison of the relative affinities of the anti-CT L A-4 single-chain antibody of the E L ISA gradient in example 3 of the present invention;
FIG. 3 is a map of an expression plasmid pTSE for anti-CT L A-4 whole antibody provided in example 3 of the present invention;
FIG. 4 is a graph showing a comparison of the binding ability of the anti-CT L A-4 monoclonal antibody to the CT L A-4 antigen in example 5 of the present invention;
FIG. 5 is a graph showing the analysis of the binding specificity of the anti-CT L A-4 whole antibody to the cell surface CT L A-4 antigen in example 6 of the present invention;
FIG. 6 is a graph comparing the activities of the anti-CT L A-4 monoclonal antibodies tested in the mixed lymphocyte reaction (M L R) in example 7 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples.
Example 1
The embodiment 1 of the invention provides a novel anti-CT L A-4 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8, and the amino acid sequence of the light chain variable region is SEQ ID NO 9 or SEQ ID NO 10.
The invention constructs a novel anti-CT L A-4 monoclonal antibody by utilizing a method for obtaining a specific antibody by utilizing a fully-synthesized ScFv single-chain phage antibody library, the fully-humanized monoclonal antibody specifically combined with a CT L A-4 antigen is obtained by utilizing phage antibody library technology for screening, and the specific biopanning method for the anti-CT L A-4 single-chain antibody is as follows:
the method comprises the following steps: a series of gene cloning methods are adopted to modify a vector pComb3 (purchased from China plasmid vector strain cell strain gene collection center) for construction and expression of a phage single-chain antibody library. The transformed vector is named pScFvDisb-s, the plasmid map of the transformed vector is shown in figure 1, and a fully synthetic phage antibody library is constructed on the basis of the vector.
Step two, using CT L A-4 as antigen coating immune tube, the antigen coating amount is 2 mug/500 mug/tube, coating overnight at 4 ℃, adding phage antibody library into immune tube for antigen-antibody combination, the phage input amount is about 109～1012After reaction at room temperature for 1 hour, PBS was usedT-PBS washed away unbound phage, eluted by 0.1M Glycine-HCl pH 2.2, and finally the eluted phage antibody solution was neutralized to about pH 7.0 using 1.5M Tris-HCl pH 8.8.
Step three: and infecting 10ml of TG1 bacterial solution growing to the logarithmic phase with the neutralized phage, standing in an incubator at 37 ℃ for 30min, taking out part of the bacterial solution, performing gradient dilution, coating on a 2YTAG plate, and calculating the output of the phage. The remaining bacterial solution was centrifuged and the supernatant discarded, and the pellet was resuspended in a small volume of medium, aspirated and plated on a 2YTAG large plate in preparation for the next round of screening.
Step four: scraping the infected and plated thallus from a large plate, inoculating the thallus to a 2YTAG liquid culture medium, shaking to logarithmic phase, adding M13 helper phage for superinfection, culturing at 28 ℃ overnight to amplify the phage, and depositing and purifying the phage by PEG6000-NaCl for the next round of screening. Three rounds of enrichment and screening of phage library are performed according to the method for later use.
Step five, screening positive clones of the single-chain antibody of the CT L A-4 phage, selecting well-separated monoclonal colonies after three rounds of screening, inoculating the colonies to a 96-hole deep-hole plate added with a 2YTAG liquid culture medium, culturing the colonies to the logarithmic growth phase under the conditions of 37 ℃ and 220rpm, and adding about 10 phage single-chain antibody clones to each hole10The helper phage M13KO7 is statically infected at 37 ℃ for 30min, centrifuged at 4000rpm and 4 ℃ for 15min, the supernatant is discarded, the phage is resuspended and precipitated by 2YTAK, cultured overnight at 28 ℃ and 220rpm, the amplified phage supernatant is absorbed for E L ISA identification, and monoclonal antibodies with higher affinity are obtained by screening, which are respectively named as N-1, N-2, N-3, N-4, N-5, N-6, N-7 and N-8, and the obtained monoclonal antibodies are sent to Sci Biotech Limited for gene sequencing to determine the monoclonal antibodies as correct antibody sequences.
After sequencing, the sequences of the 8 monoclonal antibodies screened above are as follows, and the monoclonal antibody protected by the invention is selected from any one of the following:
(N-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10.
Specifically, SEQ ID NO:1 (amino acid sequence of heavy chain variable region in N-1):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYNGNWVRQAPGKGLEWVTFISYDGLNDVKADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
2 (amino acid sequence of heavy chain variable region in N-2):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSIFKRWVRQAPGKGLEWVTFISYDGLDHINGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
3 (amino acid sequence of heavy chain variable region in N-3):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGIFSSWVRQAPGKGLEWVTFISYDSLKQKDGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
4 (amino acid sequence of heavy chain variable region in N-4):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSAFYNPWVRQAPGKGLEWVTFISYDGVYFHFGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
5 (amino acid sequence of heavy chain variable region in N-5):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGFFNRWVRQAPGKGLEWVTFISYDALKDFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGPGTLVTVSS；
6 (amino acid sequence of heavy chain variable region in N-6):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSNTLRWVRQAPGKGLEWVTFISYDSLFLDQGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
SEQ ID NO:7 (amino acid sequence of heavy chain variable region in N-7):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGIYCNWVRQAPGKGLEWVTFISYDSILNVIADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
SEQ ID NO:8 (amino acid sequence of heavy chain variable region in N-8):
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGFSICWVRQAPGKGLEWVTFISYDGLLFLIADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARRYRWWGKAMDYWGQGTLVTVSS；
9 (amino acid sequence of light chain variable region in N-1-4):
DIVMTQSPLSLPVTPGEPASISCKSSQSVIDSKGYTYLGWYLQKPGQSPQLLIYLGTQKSSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQRGHIPITFGQGTKLEIK；
10 (amino acid sequence of light chain variable region in N-5-8):
DIVMTQSPLSLPVTPGEPASISCRSSQTVFDSHGQTYLQWYLQKPGQSPQLLIYGASRQVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVWHLPITFGQGTKLEIK。
further, the monoclonal antibody also comprises a heavy chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG 4;
preferably, the heavy chain constant region is human IgG 1.
Furthermore, the monoclonal antibody is a full-length antibody or an antibody fragment, and the antibody fragment comprises one or a combination of more of Fab, F (ab)2, Fv or ScFv.
Further, the monoclonal antibody is a fully human antibody.
Example 2
The embodiment 2 of the invention also provides a polypeptide or protein, and the polypeptide or protein comprises the novel anti-CT L A-4 monoclonal antibody.
The invention also provides a polynucleotide sequence or a combination thereof, and the polynucleotide sequence or the combination thereof codes the amino acid sequence of the novel anti-CT L A-4 monoclonal antibody.
The invention also provides a recombinant DNA expression vector comprising a polynucleotide sequence or a combination.
The invention also provides a host cell for transfecting the recombinant DNA expression vector, wherein the host cell comprises prokaryotic cells, yeast cells, insect cells or mammalian cells.
Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
The invention further provides a medicine or a medicine composition, and the medicine or the medicine composition comprises the novel anti-CT L A-4 monoclonal antibody.
The invention further provides application of the novel anti-CT L A-4 monoclonal antibody in preparing a medicament for treating cancer diseases, wherein the cancer diseases comprise melanoma, prostatic cancer, bladder cancer, colorectal cancer, gastrointestinal cancer or liver cancer.
Example 3 gradient dilution of phage E L ISA comparison of affinity of anti-CT L A-4 monoclonal antibody
The monoclonal antibodies of 8 strains (N-1, N-2, N-3, N-4, N-5, N-6, N-7 and N-8) obtained in example 1 were displayed and purified by monoclonal phage, and then subjected to phage gradient dilution E L ISA test to identify affinity, and the anti-CT L A-4 monoclonal antibody provided by core patent EP2829609A1 of Yervoy, a commercial product, was selected as a positive control, as follows:
coating CT L A-4 with carbonate buffer solution of pH9.6, 100 ng/well/100. mu.l, coating overnight at 4 deg.C, washing three times with PBST, diluting the 8 monoclonal antibodies screened in example 1 and the anti-CT L A-4 monoclonal antibody provided in patent EP2829609A1 with PBST four-fold gradient, adding 100. mu.l diluted sample to each well, standing at room temperature for 1 hour, washing E L ISA plate with PBST, adding the HRP-anti-M13 monoclonal antibody diluted with PBST to E L ISA plate,standing at room temperature for 1 h. The TMB color development kit develops color at room temperature for 10 minutes. With 2M H2SO4After termination, 450nm/630nm was read.
As shown in FIG. 2, the selected 8 different monoclonal antibodies can bind to CT L A-4, and compared with the anti-CT L A-4 monoclonal antibody provided by patent EP2829609A1, the monoclonal antibody provided by the invention has stronger binding ability to CT L A-4 and higher affinity.
Example 4 preparation of anti-CT L A-4 Whole antibody
The heavy chain VH and light chain Vkappa genes of the 8 monoclonal antibodies selected in example 1 were cloned into vector pTSE (shown in FIG. 3) containing heavy chain and light chain constant region genes, respectively, the heavy chain constant region is human IgG1 constant region (amino acid sequence is shown in SEQ ID NO:11), the light chain constant region is kappa chain constant region (amino acid sequence is shown in SEQ ID NO:12), the pTSE vector structure is shown in FIG. 3, and the preparation process is described in page 3 [0019] of the CN103525868A specification.
11 (constant region sequence of human IgG 1):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG；
SEQ ID NO:12 (light chain constant region sequence of kappa chain):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
HEK293E cells were transiently transfected for whole antibody expression, and whole antibody proteins of 8 monoclonal antibodies were obtained by protein A affinity column purification using AKTA instrument, while protein concentration was determined using BCA kit.
EXAMPLE 5 binding of Whole antibody to CT L A-4
Coating CT L A-4 with carbonate buffer pH9.6 at 100 ng/well/100. mu.l, coating overnight at 4 deg.C, and coating with 300. mu.l/well PWashing with BST three times, adding whole antibody with different dilution concentrations and anti-CT L A-4 monoclonal antibody provided by patent EP2829609A1, wherein the initial highest concentration of all antibodies is 10 μ g/m L, diluting with 5 times, performing 8 gradients on each whole antibody, incubating at 37 deg.C for 2-3h, washing with 300 μ l/well PBST five times, adding anti-human IgG-HRP diluted with PBST 1: 5000, incubating at 37 deg.C for 1h, washing with 300 μ l/well PBST eight times, developing with TMB developing kit, developing at 100 μ l/well, developing at room temperature for 10min, and then developing with 2M H2SO4 terminates. Readings were taken at 450nm/630nm and the corresponding EC50 values were calculated as follows:
| NAME | N-1 | N-2 | N-3 | N-4 | N-5 | N-6 | N-7 | N-8 | EP2829609A1 |
| EC50(ng/ml) | 162.7 | 109.6 | 263.6 | 92.24 | 68.86 | 127.4 | 86.53 | 164.4 | 689.0 |
through the data and as shown in FIG. 4, the selected full antibodies of 8 different monoclonal antibodies can be combined with CT L A-4, the EC50 values of the 8 different monoclonal antibodies provided by the invention are obviously lower than that of the full antibody of the anti-CT L A-4 monoclonal antibody provided by the patent EP2829609A1, which shows that the monoclonal antibody provided by the invention is higher in binding affinity with CT L A-4 and has better activity, and in addition, as can be seen from FIG. 4, the full antibody of N-5 in the 8 full antibodies is the best in activity and the EC50 value is the lowest, which shows that the full antibody is the best in binding ability with CT L A-4, the highest in affinity and the best in activity.
Example 6 analysis of binding specificity of Whole antibody to cell surface CT L A-4
The invention adopts CHO cells over-expressing CT L A-4 antigen to detect the binding condition of different anti-CT L A-4 monoclonal antibodies and cell surface CT L A-4, uses human IgG (hIgG) as isotype control, selects the whole antibody of anti-CT L A-4 monoclonal antibody provided by core patent EP2829609A1 of the commercial product Yervoy as positive control, and adopts the concrete method that centrifugally collects cells, simultaneously dilutes various antibodies, the highest concentration is 20 mu g/m L, 4 times of gradient dilution, washes the collected cells with PBS + 1% BSA for three times, then adds PBS + 1% BSA to resuspend the cells, then spreads the cells in a 96-well plate, and each well is 1 × 105Adding 100 μ l of diluted anti-CT L A-4 monoclonal antibody and positive control and isotype control into each cell, incubating at room temperature for 1 hr, centrifuging to remove supernatant, washing the cells with PBS for three times, resuspending the cells with diluted Alexa 488-labeled anti-human IgG FC antibody solution, incubating at room temperature in the dark for 1 hr, washing with PBS for three times, resuspending with 100ul PBS, and flowingThe fluorescence intensity was measured by a cytometer. The results were analyzed using Graphpad Prism and the corresponding EC50 values were calculated as follows:
| NAME | N-1 | N-2 | N-3 | N-4 | N-5 | N-6 | N-7 | N-8 | EP2829609A1 | hIgG |
| EC50(ng/ml) | 7.095 | 8.946 | 8.963 | 11.12 | 6.118 | 7.047 | 7.925 | 6.492 | 92.65 | —— |
based on the above data and as shown in fig. 5, the EC50 values of the 8 full antibodies prepared in example 4 of the present invention are all significantly lower than the full antibody against CT L a-4 monoclonal antibody provided in patent EP2829609a1, which indicates that the 8 full antibodies provided in the present invention can bind to CT L a-4 expressed by cells, and the EC50 value of the N-5 full antibody in the 8 full antibodies is the lowest, indicating that the full antibody specifically binds to CT L a-4 antigen best, the affinity is the strongest, and the activity is the best.
Example 7 Mixed lymphocyte reaction (M L R) testing of anti-CT L A-4 monoclonal antibody Activity
Separating PBMC from fresh peripheral blood by density gradient centrifugation, and sorting CD14 with magnetic beads+Culturing CD14 in culture medium of 20ng/m L GM-CSF and 10ng/m L I L-4+And (3) changing the liquid every 2 days, inducing the DC into dendritic DC cells after 7-10 days, adding 25ng/m L TNF- α to induce the DC into mature DC cells two days before the DC is used, collecting the mature DC cells, and preparing the DC cells into the DC cells with the cell density of 1x105Cell suspension of L/m CD4 was separated from fresh PBMC by magnetic bead separation+T cells were counted to make the cell density 1x106Cell suspension of L pieces/m CD4+Taking 100ul of T cells and DC cells respectively, and mixing the T cells and the DC cells according to the proportion of 10: 1 into 96-well plates.
8 strains of anti-CT L A-4 whole antibody prepared in example 4, anti-CT L A-4 whole antibody provided in patent EP2829609A1 as a positive control and human IgG (hIgG) as an isotype control were diluted in 4-fold gradients, each of which was set at 6 gradients, 50ul each was added to a 96-well plate, and after 5 days, cck8 tested CD4+T cells were propagated, read at 450nm, and corresponding EC50 values were calculated as follows:
| NAME | N-1 | N-2 | N-3 | N-4 | N-5 | N-6 | N-7 | N-8 | EP2829609A1 | hIgG |
| EC50(mol/L) | 7.343E-09 | 8.308E-09 | 1.418E-08 | 1.538E-08 | 4.946E-09 | 6.67E-09 | 6.307E-09 | 7.318E-09 | 8.513E-08 | —— |
through the data and as shown in FIG. 6, the EC50 values of 8 different anti-CT L A-4 full antibodies screened by the invention are all obviously lower than that of the anti-CT L A-4 full antibody provided by the patent EP2829609A1, which shows that the activity of the anti-CT L A-4 full antibody provided by the invention is all higher than that of the anti-CT L A-4 full antibody provided by the patent EP2829609A1, which shows that the activity of the anti-CT L A-4 monoclonal antibody provided by the invention is better and the affinity is stronger, and in addition, as can be seen from FIG. 6, the N-5 activity is the highest in the 8 monoclonal antibodies screened by the invention.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Sequence listing
<110> Beijing Oriental Baitai Biotechnology Ltd
<120> novel anti-CT L A-4 monoclonal antibody and application thereof
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Gly Thr Leu Val Thr Val Ser Ser
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<213> Artificial sequence (Homo sapiens)
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Gly Thr Leu Val Thr Val Ser Ser
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Phe
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100 105 110
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1 5 10 15
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20 25 30
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<210>10
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Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Val Phe Asp Ser
20 25 30
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100 105 110
<210>11
<211>329
<212>PRT
<213> Artificial sequence (Homo sapiens)
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1 5 10 15
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20 25 30
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35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
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Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
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305 310315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
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<210>12
<211>107
<212>PRT
<213> Artificial sequence (Homo sapiens)
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
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Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
Claims (11)
1. An anti-CT L a-4 monoclonal antibody, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the monoclonal antibody is selected from any one of the following:
(N-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 9;
(N-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10;
(N-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10.
2. The anti-CT L a-4 monoclonal antibody of claim 1, further comprising a heavy chain constant region that is one of human IgG1, IgG2, IgG3, IgG 4.
3. The anti-CT L a-4 monoclonal antibody of claim 2, wherein the heavy chain constant region is human IgG 1.
4. The anti-CT L a-4 monoclonal antibody of claim 1, wherein the monoclonal antibody is a full-length antibody or an antibody fragment comprising one or a combination of Fab, F (ab)2, Fv or ScFv.
5. A protein which is the anti-CT L A-4 monoclonal antibody of any one of claims 1-4.
6. A polynucleotide molecule encoding the anti-CT L a-4 monoclonal antibody of any one of claims 1-4.
7. A recombinant DNA expression vector comprising the polynucleotide molecule of claim 6.
8. A host cell transfected with the recombinant DNA expression vector of claim 7, wherein said host cell comprises a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell.
9. A host cell transfected with the recombinant DNA expression vector of claim 8, wherein said host cell is a mammalian cell, and wherein said mammalian cell is a HEK293E cell, a CHO cell, or a NS0 cell.
10. A medicament comprising the anti-CT L a-4 monoclonal antibody of any one of claims 1-4.
11. Use of an anti-CT L a-4 monoclonal antibody of any one of claims 1-4 in the manufacture of a medicament for the treatment of a cancer disease including melanoma, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer or liver cancer.
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Address after: 406, room 1, building 2, 100176 Jingdong street, Beijing economic and Technological Development Zone, Beijing, Daxing District Patentee after: Beijing Dongfang Baitai Biotechnology Co., Ltd Address before: 406, room 1, building 2, 100176 Jingdong street, Beijing economic and Technological Development Zone, Beijing, Daxing District Patentee before: Beijing Dongfang Biotech Co.,Ltd. |
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